The present disclosure relates to antibodies that bind CD47, including that of humans and other mammalian species, and their use in treating conditions and disorders, such as ischemia-reperfusion injury (IRI) and cancers, mediated by this receptor.
CD47 is a cell surface receptor comprised of an extracellular IgV set domain, a 5 membrane spanning transmembrane domain, and a cytoplasmic tail that is alternatively spliced. Two ligands bind CD47: thrombospondin-1 (TSP1), and signal inhibitory receptor protein alpha (SIRPalpha). TSP1 binding to CD47 activates the heterotrimeric G protein Gi, which leads to suppression of intracellular cyclic AMP (cAMP) levels. In addition, the TSP1-CD47 pathway opposes the beneficial effects of the nitric oxide pathway in all vascular cells. The nitric oxide (NO) pathway consists of any of three nitric oxide synthase enzymes (NOS I, NOS II and NOS III) that generate bioactive gas NO using arginine as a substrate. NO can act within the cell in which it is produced, or in neighboring cells, to activate the enzyme soluble guanylyl cyclase that produces the messenger molecule cyclic GMP (cGMP). The proper functioning of the NO-cGMP pathway is essential for protecting the cardiovascular system against stresses including, but not limited to, those resulting from wounding, inflammation, hypertension, metabolic syndrome, ischemia, and ischemia-reperfusion injury (IRI). In the context of these cellular stresses, the inhibition of the NO-cGMP pathway by the TSP1-CD47 system exacerbates the effects of stress. This is a particular problem in the cardiovascular system where both cGMP and cAMP play important protective roles. There are many cases in which ischemia and reperfusion injury cause or contribute to disease, trauma, and poor outcomes of surgical procedures.
SIRPalpha is expressed on hematopoietic cells, including macrophages and dendritic cells. When it engages CD47 on a potential phagocytic target cell, phagocytosis is slowed or prevented. The CD47-SIRPalpha interaction effectively sends a “don't eat me” signal to the phagocyte. Thus, blocking the SIRPalpha-CD47 interaction with a monoclonal antibody in this therapeutic context can provide an effective anti-cancer therapy by promoting, i.e., increasing, the uptake and clearance of cancer cells by the host's immune system by increasing phagocytosis. This mechanism is effective in leukemias, lymphomas, and many types of solid tumors.
U.S. Pat. No. 8,236,313 contemplates antibodies that could be useful in the field of ischemia and blood flow to reverse and/or prevent tissue ischemia and related and associated tissue and cell damage, including antibodies that block CD47.
U.S. Pat. No. 8,101,719 discloses humanized antibodies that bind to CD47 for use in treating hematological disorders. Objects of the invention include humanized anti-CD47 antibodies and small antibody fragments exhibiting reduced antigenicity while retaining their CD47 binding activity and apoptosis-inducing activity. Such antibodies and small fragments are contemplated for use in treating hematological disorders such as various types of leukemias, malignant lymphoma, aplastic anemia, myelodysplastic syndromes, and polycythemia vera.
PCT International Publication WO 2011/143624 discloses chimeric and humanized anti-CD47 monoclonal antibodies for use as reagents for the diagnosis and immunotherapy of diseases associated with CD47 in humans, particularly in cancer therapy, for example to increase phagocytosis of cancer cells expressing CD47. Preferred antibodies are non-activating, i.e., block ligand binding, but do not signal. Disclosed humanized B6H12 and 5F9 antibodies bound soluble human CD47; B6H12 also bound human CD47 on the surface of human CD47-transfected YB2/0 cells. Humanized B6H12 and 5F9 antibodies enabled phagocytosis of CFSE-labeled HL-60 cells by mouse bone marrow- or peripheral blood-derived macrophages in vitro, respectively. Humanized B6H12 utilized human VH-3-7 and VK3-11 frameworks.
U.S. 2013/0142786 discloses non-activating anti-CD47 antibodies that increase the phagocytosis of CD47 expressing cells and these humanized or chimeric anti-CD47 antibodies can be used for therapeutic purposes, particularly in cancer therapy. Amino acid sequences of murine and humanized mAb B6H12, 5F9, and 8B6 heavy and light chain variable regions are disclosed.
Han et al. (2000) J. Biol. Chem. 275(48):37984-37992 discloses the production of mouse anti-CD47 monoclonal antibodies 400 (IgG2b), 410 (IgG1), 420 (IgG2a), 430 (IgG2a), 440 (IgG1), 450 (IgG2a), 460 (IgG1), 470 (IgG2a), and 480 (IgG1) generated by immunizing CD47-deficient mice with the extracellular domain of human CD47. No amino acid sequences of these antibodies, or their CDRs, are disclosed. Of these nine mAbs, three blocked macrophage fusion: 430, 450, and 470. Han et al. discloses no data either demonstrating or suggesting that CD47 plays a role in ischemia-reperfusion injury, autoimmune or inflammatory diseases, or cancer.
PCT International Publication WO 2013/119714 discloses anti-CD47 antibodies that do not cause a significant level of hemagglutination of human red blood cells.
There exists a need for antibodies to human CD47 that selectively block the binding of TSP1 to CD47 to promote the beneficial effects of nitric oxide-cGMP signaling and cAMP signaling in the cardiovascular system in settings in which IRI plays a role in pathogenesis. These situations/diseases include organ transplantation, acute kidney injury, cardiopulmonary bypass surgery, pulmonary hypertension, sickle cell disease, myocardial infarction, stroke, surgical resections and reconstructive surgery, reattachment of digits/body parts, skin grafting, and trauma. There is also a need for antibodies that block the binding of SIRPalpha to CD47, thus providing novel anti-cancer therapies.
Antibody compounds of the present disclosure meet these needs. They bind to epitopes in the extracellular IgV domain of CD47, variously inhibiting the binding of SIRPalpha and TSP1 to CD47 and receptor activation. Antibodies that block TSP1 and SIRPalpha binding should be therapeutically useful in preventing, treating, or reducing many forms of IRI and treating cancers. Antibodies that block SIRPalpha binding promote phagocytosis of cancer cells. In view of these properties, SIRPalpha blocking antibody compounds should be therapeutically useful in treating a variety of cancers, including hematological cancers and solid tumors.